Similar Datasets
Project description:Evaluting HLA loss of heterozygosity, a key immune escape mechanism, using NGS and immunopeptidomics.
2021-11-04 | PXD022323 | Pride
Project description:HLA-B*51 allele with p.N63S
| PRJEB111377 | ENA
Project description:Human leukocyte antigen (HLA) - B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are two strong predisposing genetic factors to Behçet's disease (BD). Previous studies have focused on two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry. The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2. This unconventional group of HLA-B*51:01-bound peptides was enriched for 8-mers (with fewer 9-mers) compared to Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides with the exception of the less abundant Leucine at position Ω. Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type - dependent manner. The HLA-B*51:01 peptidome includes a surprisingly high proportion of unconventional non-Pro/Ala2 peptides which are increased by ERAP1 silencing, mimicking the loss-of-function BD risk variant.
2020-03-18 | PXD013064 | Pride
Project description:HLA-B*51, the main risk factor for Behçet’s disease (BD), binds two main subpeptidomes consisting of low affinity peptides with Ala as their second amino acid and higher affinity peptides with Pro at this position. A low activity variant of ERAP1, Hap10, is uniquely associated with BD susceptibility in epistasis with HLA-B*51. The peptidome presented by HLA-B*51:08 in a cell line expressing Hap10 was compared with the HLA-B*51:01 peptidome from a cell line expressing higher activity variants of ERAP1. The differences due to ERAP1 could be clearly distinguished from those due to subtype polymorphism. The latter should not affect disease susceptibility, since both HLA-B*51 subtypes are associated with BD. In the lower activity, BD-associated, ERAP1 context longer peptides were generated, the Pro2 subpeptidome was significantly reduced, and the Ala2 subpeptidome was correspondingly increased and showed a higher frequency of ERAP1-susceptible P1 residues. These effects are readily explained by the low activity of the disease-associated Hap10 variant, and together resulted in a significantly altered HLA-B*51 peptidome of lower affinity. The differences between both B*51 subtypes affected residue usage at various internal positions of the peptide ligands, including P3, where B*51:01 showed preference for aromatic residues whereas B*51:08 preferred smaller and polar ones. The profound effects of the BD-associated Hap10 haplotype on the nature and affinity of HLA-B*51/peptide complexes could significantly alter T-cell and NK cell recognition, providing a basis for the joint association of ERAP1 and HLA-B*51 to BD.
2018-10-24 | PXD006215 | Pride
Project description:The endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 trim peptides to be loaded onto HLA molecules, including the main risk factor for Behçet’s disease HLA-B*51. ERAP1 is also a risk factor among HLA-B*51-positive individuals, whereas no association is known with ERAP2. This study addressed the mutual relationships between both enzymes in the processing of an HLA-bound peptidome, interrogating their differential association with Behçet’s disease. CRISPR/Cas9 was used to generate knock outs of ERAP1, ERAP2 or both from transfectant 721.221-HLA-B*51:01 cells. The surface expression of HLA-B*51 was reduced in all cases. The effects of depleting each or both enzymes on the B*51:01 peptidome were analyzed by quantitative label-free mass spectrometry. Substantial quantitative alterations of peptide length, subpeptidome balance, N-terminal residue usage, affinity and presentation of non-canonical ligands were observed. These effects were often different in the presence or absence of the other enzyme, revealing their mutual dependency. In the absence of ERAP1, ERAP2 showed similar and significant processing of B*51:01 ligands, indicating functional redundancy. The high overlap between the peptidomes of wildtype and double KO cells indicates that a large majority of B*51:01 ligands are present in the ER even in the absence of ERAP1/ERAP2. These results indicate that both enzymes have distinct, but complementary and partially redundant effects on the B*51:01 peptidome, leading to its optimization and maximal surface expression. The distinct effects of both enzymes on the HLA-B*51 peptidome provide a basis for their differential association with Behçet’s disease and suggest a pathogenetic role of the B*51:01 peptidome.
2019-05-20 | PXD012348 | Pride